Evidence that human and equine erythrocytes could have significant roles in the transport and delivery of amino acids to organs and tissues.

Erythrocytes have a well-defined role in the gaseous exchange of oxygen and carbon dioxide in the mammalian body. The erythrocytes can contain more than half of the free amino acids present in whole blood. Based on measures showing that venous erythrocyte levels of amino acids are much less than arterial erythrocyte levels, it has previously been proposed that erythrocytes also play a role in the delivery of amino acids to tissues in the body. This role has been dismissed because it has been assumed that to act as an amino acid transport vehicle, the erythrocytes should release their entire amino acid content in the capillary beds at the target tissues with kinetic studies showing that this would take too long to achieve. This investigation set out to investigate whether the equine erythrocytes could rapidly take up and release smaller packages of amino acids when exposed to high or low external concentrations of amino acids, because it seemed very unlikely that cells would be able to release all of their amino acids without serious impacts on osmotic balance. Freshly prepared erythrocytes were placed in alternating solutions of high and low amino acid concentrations in PBS to assess the capacities of these cells to rapidly take up and release amino acids depending on the nature of the external environment. It was found that amino acids were rapidly taken up and released in small quantities in each cycle representing 15% of their total load in equine erythrocytes and 16% in human erythrocytes. The capacity for rapid uptake/release of amino acids by equine and human erythrocytes provided evidence to support the theory that mammalian erythrocytes have a significant role in transport of amino acids from the liver to tissues, muscles and organs.

Modelling of protein turnover provides insight for metabolic demands on those specific amino acids utilised at disproportionately faster rates than other amino acids.

The nitrogen balance is regulated by factors such as diet, physical activity, age, pathogenic challenges, and climatic conditions. A paradigm was developed from published recommended rates of protein intake (g/kg/day) with corresponding rates of endogenous protein turnover and excretion, to extrapolate amino acid balances under various conditions. The average proportions of amino acids in the ingested proteins representing a well-balanced diet were used to assess intake and an average human composition profile from five major high-turnover proteins in the body to assess endogenous protein turnover. The amino acid excretion profiles for urine and sweat were constructed for males and females from published data. The model calculated the nitrogen balances for a range of amino acids to determine the amino acid requirements to support daily exertion. Histidine, serine, glycine, and ornithine were in negative balances in males and females and this potential deficit was greater in the higher body-mass ranges. Conversely, leucine, isoleucine, and valine were conserved during nitrogen flux and resulted in positive balances. The model was run under a scenario of high demand for the synthesis of IgG during a response to an infectious challenge which indicated that these were increased requirements for tyrosine, threonine, and valine. It was concluded that these amino acids represent points of limitation to anabolic metabolism by restriction of their supply at critical times of demand. This would especially occur under conditions of fitness training, maintaining intensive exercise regimes, facilitating responses to pathogenic challenge, or recovery from injury.

Sex differences in amino acids lost via sweating could lead to differential susceptibilities to disturbances in nitrogen balance and collagen turnover.

Fluid collected during sweating is enriched with amino acids derived from the skin's natural moisturising factors and has been termed "faux" sweat. Little is known about sex differences in sweat amino acid composition or whether faux sweat amino acid losses affect nitrogen balance. Faux sweat collected by healthy adults (n = 47) after exercise, and at rest by chronic fatigue patients, was analysed for amino acid composition. Healthy females had higher total amino acid concentrations in sweat (10.5 ± 1.2 mM) compared with healthy males (6.9 ± 0.9 mM). Females had higher levels of 13 amino acids in sweat including serine, alanine and glycine. Higher hydroxyproline and proline levels suggested greater collagen turnover in females. Modelling indicated that with conservative levels of exercise, amino acid losses in females via faux sweat were triple than those predicted for urine, whereas in males they were double. It was concluded that females were more susceptible to key amino acid loss during exercise and/or hot conditions. Females reporting chronic fatigue had higher levels of methionine in faux sweat than healthy females. Males reporting chronic fatigue had higher levels of numerous amino acids in faux sweat compared to healthy males. Higher amino acid loss in faux sweat associated with chronic fatigue could contribute to a hypometabolic state. Depending on activity levels, climatic conditions and gender, amino acid losses in sweat and skin leachate could influence daily protein turnover where periods of continuously high turnover could lead to a negative net nitrogen balance.

Diverse characteristics of the urinary excretion of amino acids in humans and the use of amino acid supplementation to reduce fatigue and sub-health in adults.

BACKGROUND: The excretion of amino acids in urine represents an important avenue for the loss of key nutrients. Some amino acids such as glycine and histidine are lost in higher abundance than others. These two amino acids perform important physiological functions and are required for the synthesis of key proteins such as haemoglobin and collagen. METHODS: Stage 1 of this study involved healthy subjects (n = 151) who provided first of the morning urine samples and completed symptom questionnaires. Urine was analysed for amino acid composition by gas chromatography. Stage 2 involved a subset of the initial cohort (n = 37) who completed a 30 day trial of an amino acid supplement and subsequent symptom profile evaluation. RESULTS: Analyses of urinary amino acid profiles revealed that three groups could be objectively defined from the 151 participants using k-means clustering. The amino acid profiles were significantly different between each of the clusters (Wilks' Lambda = 0.13, p < 0.0001). Cluster 1 had the highest loss of amino acids with histidine being the most abundant component. Cluster 2 had glycine present as the most abundant urinary amino acid and cluster 3 had equivalent abundances of glycine and histidine. Strong associations were observed between urinary proline concentrations and fatigue/pain scores (r = .56 to .83) for females in cluster 1, with several other differential sets of associations observed for the other clusters. CONCLUSIONS: Different phenotypic subsets exist in the population based on amino acid excretion characteristics found in urine. Provision of the supplement resulted in significant improvements in reported fatigue and sleep for 81% of the trial cohort with all females reporting improvements in fatigue. TRIAL REGISTRATION: The study was registered on the 18th April 2011 with the Australian New Zealand Clinical Trials Registry ( ACTRN12611000403932 ).

Health characteristics of inland waterway merchant marine captains and pilots.

BACKGROUND: Most published studies on seafarer health have focused on patterns of mortality, injury and communicable diseases. Little information is available regarding lifestyle-related cardio-metabolic disease in maritime populations. AIMS: To describe health characteristics of a population of US inland waterway merchant marine captains and pilots. METHODS: A cross-sectional study of the health characteristics of mariners required to complete the United States Coast Guard physical assessment at a regional medical centre from 2003-10. Variables collected included self-reported smoking status, body mass index, fasting lipids, glucose and triglyceride levels, blood pressure and treadmill time and maximal oxygen uptake as measured using the Bruce Protocol. Major medical conditions related to lifestyle and risk for metabolic syndrome were also assessed. RESULTS: There were 388 participants. The study population had high prevalence of obesity (61%), smoking (41%), high triglycerides (42%), low HDL cholesterol (47%), high blood pressure (42%), high fasting glucose (22%) and three or more features of the metabolic syndrome (39%). CONCLUSIONS: This population exhibited a high prevalence of chronic disease risk factors and could potentially benefit from health promotion programmes aimed at improving health and fitness.

Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia.

OBJECTIVE: To detect Anaplasma platys and Babesia canis vogeli infection, using polymerase chain reaction (PCR)-based assays, in free-roaming dogs associated with eight Aboriginal communities in remote areas of Australia and to determine the impact of infection through the assessment of platelet numbers. PROCEDURES: Blood samples from 215 dogs were screened by PCR for A platys and B canis vogeli using established genus-specific DNA primers for the 16S and 18S rRNA genes respectively. Both A platys DNA and B canis vogeli DNA were confirmed from the screening PCR either by sequencing or by the use of species-specific primers. Peripheral blood films from 92 of the 215 dogs were used to estimate platelet numbers through an indirect method. RESULTS: Of 215 dogs, 69 (32%) were positive for A platys, 22 (10%) for B canis vogeli and 24 (11%) for both. The two organisms were detected singularly and as coinfection in all communities. For the 92 dogs in which peripheral blood films were examined, the mean estimated platelet counts for the non-infected dogs was 318 x 10(9)/L, those infected with A platys alone was 256 x 10(9)/L, those with B canis vogeli alone was 276 x 10(9)/L and those infected with both parasites was 169 x 10(9)/L. In young dogs, infection produced significantly decreased mean platelet counts when compared to uninfected dogs. Thrombocytopenia (< 200 x 10(9)/L) was detected in 18 (51%) dogs infected with A platys alone, 3 (33%) dogs infected with B canis vogeli alone, 13 (72%) dogs coinfected, and 8 (27%) uninfected dogs. CONCLUSIONS: A platys and B canis vogeli infection, either singularly or together, was widespread in free roaming dogs associated with remote Aboriginal communities in the Northern Territory and north-western New South Wales. Moreover, both A platys and B canis vogeli infections were associated with a reduction in mean platelet numbers in dog populations, particularly in young dogs. The fact that 51% of dogs infected with A platys alone and 72% dogs coinfected were thrombocytopenic compared to 27% of uninfected dogs suggests that the organism alone or in combination with B canis vogeli has the potential to cause thrombocytopenia and perhaps contribute to a clinical bleeding disorder in infected dogs.

Ovine placental eluate immunoglobulins recognise isologous and third party acid-treated trophoblast microvesicle antigens in vitro.

Placental microvesicles were prepared from ovine placentae and immunoglobulins eluted with 0.5 M glycine buffer pH 2.5. The ability of eluate immunoglobulins to re-associate with isologous (self) and third party acidified microvesicles was tested by ELISA. Ovine placental immunoglobulins re-associated with isologous and third party acidified microvesicles suggesting that at least 2 types of antigenic epitopes I and II maybe expressed on the ovine placentae. Type I antigens may be present on placentae of all ovines while type II epitopes may be paternally derived, hence unique to each pregnancy. Analysis by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27 kDa, respectively, together giving a relative molecular weight of 158 kDa. Results suggest that immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the trophoblast, possibly defining a mechanism by which the foetus evades maternal immunological rejection.

Esterase activity associated with secretory IgA and free secretory component preparations from human milk.

Secretory immunoglobulin A (IgA) and free secretory component were purified from human milk using methods involving (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography. It was found that the preparations, although apparently pure by conventional criteria based on immunoelectrophoresis, zone electrophoresis, or ultracentrifugation, were consistently contaminated by two different esterases. These enzymes could apparently not be removed by further ion-exchange chromatography and/or gel filtration. They could, however, be eliminated by passage on an immunoabsorbent made from anti-free secretory component antiserum.

Immunoglobulin G bound to ovine placenta is eluted by surgical cannulation and acid perfusion in situ.

OBJECTIVE: To elute placental bound immunoglobulin G (IgG) in situ. DESIGN: Laboratory based experimentation. SETTING: Biological Sciences Department, The University of Newcastle Australia and the Department of Biochemistry, University of Nairobi, Kenya. SUBJECTS: Twelve pregnant ewes 10 to 15 days before the onset of natural parturition. RESULTS: Placental eluates were rich in IgG, and IgG2. The relative molecular weight of placental IgG was estimated at 158kDa by gel filtration chromatography. Analysis of eluate by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27kDa respectively together giving a relative molecular weight of 168kDa. CONCLUSION: Placental bound IgG may be crucial in immunology of pregnancy and together with the cognate antigen thereof may be useful as models for the study of maternal-fetal interaction in human pregnancy and in the development of experimental immunotherapy to immunologically compromised pregnancies in humans and livestock.

Human placental immunoglobulins show unique re-association patterns with isologous and third party acid treated trophoblast microvesicles in vitro.

OBJECTIVE: To study re-association pattern of human placental eluate immunoglobulins with acid treated isologous and third party trophoblast derived placental microvesicles. DESIGN: Laboratory based experimentation. SETTING: Biological Sciences Department and Discipline for Reproductive Medicine University of Newcastle, Australia and the Department of Biochemistry, University of Nairobi, Kenya. RESULTS: Placental eluate immunoglobulins re-associated with isologous and third party acidified microvesicles in three distinct patterns. I: eluate immunoglobulins re-associated more strongly with isologous and third party acid treated placental microvesicles, II: eluate immunoglobulins re-associated strongly with isologous but weakly with third party acid treated placental microvesicles, III: eluate immunoglobulins did not show preferential re-association with isologous and third party acid treated placental microvesicles. CONCLUSION: Two types of antigenic epitopes I and II may be expressed on the human placentae. Type I antigens may be present on all human placentae while type II epitopes may be paternally derived hence unique to each pregnancy. Also, immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the human placental trophoblast.

The clinical relevance of alloantibody in liver transplantation.

The transplanted liver appears resistant to antibody-mediated injury compared to other transplanted organs such as kidney or heart. However, a growing number of reports suggest that alloantibody to the liver is associated with poorer outcomes. The data surrounding this field are unclear, and their interpretation remains controversial. Mechanistically, there is not a clear explanation for the liver's resistance to antibody-mediated injury, and the pathological criteria for antibody-mediated rejection (AMR) remain ill-defined. Furthermore, treatment of AMR is non-uniform. The field would benefit from better outcome data based on measurement of antibody at the time of transplantation and at the time of rejection. Consensus opinion regarding antibody and the liver might emerge with better standardization of antibody measurement and pathological definition of AMR.

Effects of EPF treatment in human mononuclear cells.

Early Pregnancy Factor (EPF) is one of the earliest pregnancy associated signals, communicating the ensuing pregnancy to the maternal organism. Data published by others on the mouse suggest that EPF bound to spleen cells causes the release of two H2-restricted "suppressor factors" responsible for the rosette inhibiting activity of EPF in the rosette inhibition test. Using human material, we were able to detect the release of a second entity from mononuclear cells that is able to suppress rosette formation in the human rosette inhibition test. In an attempt to show an intracellular EPF effect in the target cell, cytosolic free calcium concentrations were measured in EPF-treated mononuclear cells from peripheral blood. Our findings did not, however, show any changes of intracellular free Ca(2+)-concentrations under the chosen conditions.

An association of membrane-damaging toxins from coagulase-negative staphylococci and chronic orofacial muscle pain.

Forty-six patients presenting with chronic orofacial muscle pain and eight age- and sex-matched control subjects were investigated for the carriage prevalence of, and exotoxin production by, coagulase-negative staphylococci (CNS). The eight control subjects were selected from an initial group of 41 subjects on the basis of the absence of musculoskeletal symptoms. There was a significantly higher prevalence and multiple carriage of four or more strains of CNS in patients with chronic muscle pain than in control subjects (23 versus 9 isolates/10 subjects). Two of the 103 CNS isolates from patients with muscle pain and none from the control subjects produced toxic shock syndrome toxin 1 (TSST-1), suggesting that pyrogenic toxins do not significantly contribute to the aetiology of chronic muscle pain. There was a significantly higher prevalence of delta-haemolysin (41 of 114) and 'horse'-haemolysin (56 of 114) production by CNS isolates from patients with chronic muscle pain compared with those from control subjects. None of the control subjects was colonised with CNS that produced significant amount of either delta- or 'horse'-haemolysin, whereas 35 of 44 patients with chronic orofacial muscle pain were colonised with CNS that produced significant amounts of 'horse'-haemolysin, 37 that produced delta-haemolysin and 33 that produced both delta- and horse-haemolysin. This study suggests that membrane-damaging toxins, like delta- and 'horse'-haemolysin, may play a role in the aetiology of chronic orofacial muscle pain.

Muramyl peptides and the functions of sleep.

Muramyl peptides (MPs) are bacterially derived sleep factors which stimulate slow wave sleep. In the neonate, MPs are capable of inducing quiet sleep and suppressing active sleep. Given that active sleep is thought to be important for brain development during this period, the possibility that changes in the availability of MPs in the neonate may affect brain development was examined. Rat pups were given muramyl dipeptide (MDP) twice daily for the first 14 days post partum. It was hypothesised that MDP would stimulate quiet sleep at the expense of active sleep as has been shown in the young rabbit. There was no effect on neonatal levels of quiet sleep or active sleep. There was, however, a variety of effects, apparently unrelated to the sleep functions of MDP. These effects were changes in adult learning, serotonin metabolism and brain anatomy. The function of sleep in the mediation of the sleep-independent effects of MPs was examined, in particular the opposite effects of MDP on host immunity depending on the sleepiness of the host. In this light sleep does indeed serve to mediate the effects of MPs and it is speculated that many of the effects observed here may change in response to sleep levels in individuals. The notion that autonomic signalling between brain and spleen is more efficient during sleep is presented as an example of a function of sleep that may modulate the immunological effects of MPs.

An evaluation of peripheral blood platelet enumeration as a monitor of fertilization and early pregnancy.

This paper reports our data that confirm the existence of early pregnancy-associated thrombocytopenia (EPAT) in the mouse and illustrate that the phenomenon is independent of age, parity, and strain differences. This paper also provides evidence that the EPAT phenomenon is induced by a soluble factor (EPAT-factor) released by the fertilized ovum. EPAT-factor was produced in vitro by mouse embryos from the 1-cell stage to the expanded blastocyst stage. The human study involved a "blind" analysis of serum samples, collected from in vitro fertilization-treated patients, for the presence of thrombocytopenic activity. Results suggest that measurement of this thrombocytopenic activity might be useful as an index of embryo viability and might be clinically applicable for the monitoring of implantation success in in vitro fertilization programs.

Early pregnancy factor as a monitor for fertilization in women wearing intrauterine devices.

Measurement of early pregnancy factor (EPF) by the rosette inhibition test was performed on serum samples from 14 women using intrauterine devices (IUDs). Serial blood samples taken during the luteal phase of 23 cycles demonstrated in six of the cycles a transient appearance of EPF during the 6- to 8-day period following ovulation. In contrast, no EPF activity was found in sera obtained from women in whom fertilization was prevented, either by sexual abstinence or tubal ligation. These observations support the postulate that the IUD functions by the prevention of implantation of the fertilized ovum, rather than by preventing fertilization.

Validation of the rosette inhibition test for the detection of early pregnancy in women.

The authors validated use of the rosette inhibition test for the detection of early pregnancy factor (EPF) in human pregnancy, first by optimizing conditions for the assay, using known pregnant and nonpregnant sera, and second, by examining the performance of this assay in three clinical situations: a "blind" study involving coded first-trimester sera showed 80% correlation with pregnancy status; serial assay of EPF activity in sera collected from normal women attempting to conceive, correlated with human chorionic gonadotropin beta-subunit (beta-hCG) levels and pregnancy status; a longitudinal study of serial serum samples through two normal pregnancies showing the continued presence of EPF until the early third trimester in each case. It was concluded that with the rosette inhibition assay, consistent demonstration of a pregnancy-associated substance (EPF) could be obtained.

Early pregnancy factor: its role in mammalian reproduction--research review.

An immunosuppressive early pregnancy factor associated with mammalian reproduction is currently attracting considerable interest. Research into its detection, site of production, distribution, immunosuppressive property, characterization, and application is in progress in a number of laboratories. This review aims to crystallize the current research findings and to identify significant areas for further investigation and application.

Role of spermine in the cytotoxic effects of seminal plasma.

This report further characterizes the cytotoxic properties of seminal plasma and provides evidence for a role of spermine oxidation in the generation of seminal plasma cytotoxicity. Addition of spermine to lymphocyte cultures was found to result in a cytotoxic effect similar to that observed upon addition of seminal plasma. Furthermore, although seminal plasma is not cytotoxic in serum-free medium, addition of monoamine oxidase was sufficient to result in the generation of seminal plasma-associated cytotoxicity. Analysis of 73 individual seminal plasma samples indicated that all were cytotoxic, suggesting that this is an intrinsic property of seminal plasma. These results support a mechanism for seminal plasma cytotoxicity in which oxidation of spermine in seminal plasma by the amine oxidase of fetal calf serum results in generation of a cytotoxic product. It is hypothesized that this product plays a significant role in the phenomenon of seminal plasma immunosuppression. The general application of this principle to other fluids and tissues is discussed.

The relationship between the immunosuppressive and cytotoxic effects of human seminal plasma.

This report describes the cytotoxic properties of human seminal plasma and demonstrates that the inhibition of response to mitogens shown by murine lymphocytes in the presence of whole human seminal plasma can be attributed largely to an effect of seminal components on lymphocyte viability. It is hypothesised that the cytotoxic effect of seminal plasma arises as a result of the oxidation of spermine in seminal plasma by an amine oxidase enzyme present in fetal calf serum. In support of this hypothesis, it was found that seminal plasma cytotoxicity is serum dependent and is inhibited in the presence of the amine oxidase inhibitor hydroxylamine.